DNA methylation is an important epigenetic modification that serves to protect the genome from propagating mutations, and to regulate gene expression. Aberrant DNA methylation is increasingly being recognized as a major epigenetic perturbation found in many pathologic states, and has become of particular interest in the context of myelodysplastic syndrome (MDS). MDS is a state of dysregulated hematopoiesis that is characterized by increasing anemia accompanied by high marrow blast counts. MDS incidence increases with age, has substantial pathologic impact, and is sometimes a precursor to acute myeloid leukemia (AML). The relevance of aberrant DNA methylation to MDS has been supported by data on the DNA methylation profiles on hematopoietic cells from MDS patients, as well the clinical value of azacytidine and decitibine, treatments that inhibit DNA methylation and have shown at least some clinical efficacy. However, the mechanisms of DNA methylation dysregulation, as well as the manner in which perturbations of DNA methylation lead to hematologic disturbance, are opaque. Here, we will use mice as a model to study the perturbations of hematologic function that occur when specific alterations in DNA methyltransferases are induced. We hypothesize that loss of appropriate Dnmt3a or Dnmt3b expression leads to aberrant DNA methylation, altering the expression of genes that disrupt the balance between self-renewal and differentiation, ultimately leading to an impotent stem cell population unable to contribute to ongoing blood formation. We have two broad aims to explore this at functional and at molecular levels. We will determine the roles of Dnmt3s in regulation of murine hematopoietic stem cell function. We will induce deletion of the Dnmt3 genes to examine the differentiation and self-renewal properties of HSC, using stem cell purification and bone marrow transplantation. We will determine the extent to which aberrant Dnmt3 expression recapitulates an MDS-like disease in mice. Treatment of mice with DNA methylation inhibitors will reveal the functional consequences on hematopoietic progenitors and the resulting changes in methylation patterns. We will also determine the molecular consequences of loss of de novo DNA methyltransferases using global analysis of DNA methylation. These changes will be correlated with alterations in gene expression that accompany loss of Dnmt3a or Dnmt3b. We will also examine the consequences with regard to binding of additional global epigenetic regulators. These studies will allow us to determine the mode of action of the Dnmt3s in HSC, as well as to identify specific genes that are candidates for conferring the altered phenotype in Dnmt3-deleted HSCs. By analyzing the phenotypic and functional consequences of Dnmt3 loss in HSCs, we will provide the first detailed portrait of the role of Dnmt3s in any adult cell type. These data will offer insights into the role of DNA methylation alterations in pathologic states, particularly MDS. A deeper understanding of how DNA methylation regulates stem cell self-renewal and differentiation, and identification of the key genes that mediate the effects, may offer new targets for future therapeutic development.